Techniques that Revealed the Network of the Circadian Clock of Drosophila

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[21] Techniques that Revealed the Network of the Circadian Clock of Drosophila By Charlotte Helfrich-Fo¨rster Abstract

The techniques are reviewed that revealed the neuronal network of the circadian clock in the brain of the fruit fly as well as the function and localization of peripheral oscillators. Three principal techniques helped characterize the circadian clock network of Drosophila consisting of pacemaker centers in the brain and oscillators in peripheral tissues: (1) Immunolabeling with antibodies raised against specific clock proteins detected the tissues and cells that express the clock proteins, revealed the subcellular localization of clock molecules, and illuminated their abundance at different time points during the day; (2) reporter genes unraveled the network of clock neurons and reported the circadian cycling of the clock genes in vivo; and (3) genetic manipulations of clock gene expression elucidated the function of specific clock genes and clock cells. These techniques and the results gained by them are reviewed briefly. Immunocytochemistry

Immunocytochemistry relies on the specific binding of antibodies to the antigens (here clock molecules) they were raised against. Immunolabeling of clock molecules has been performed successfully on whole mounts of the central nervous system and on cryostat sections of the head or body of flies and can be regarded as a standard method established in most circadian laboratories. Antibodies against the clock molecules PERIOD (PER) and TIMELESS (TIM) revealed a broad distribution of both proteins within and outside the central nervous system, including sensory cells in the compound eyes and ocelli, the antennae and bristles on body and wings, cells of the reproductive system, cells of the Malphigian tubules, the prothoracic glands, the gut, etc. (reviewed by Hall, 1998; Helfrich-Fo¨rster, 2002). Except for the ovaries, the clock proteins varied cyclically in their abundance in all cells, demonstrating the molecular cycling of the clock. Immunostaining was highest at the end of the night and lowest at the end of the day. Furthermore, the clock proteins were merely cytoplasmatic in the middle of the night and entirely nuclear at the end of the night,

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whereby slight differences in the timing were observed between both proteins (Shafer et al., 2002). In the brain, which is relevant for behavioral rhythmicity, PER and TIM are found in many glia cells and in a few neurons named lateral neurons (LN) and dorsal neurons (DN) according to their position in the brain (Ewer et al., 1992; Frisch et al., 1994; Siwicki et al., 1988; Zerr et al., 1990; Fig. 1A). The LN and DN can each be subdivided into three cell clusters (Fig. 1A): LN cells in the anterior brain consisting of a more dorsally located cluster of 5–8 cells—the LNd—and two ventrally located cell clusters that differ in size [4–6 large LNv (l-LNv) and 5 small LNv (s-LNv)]. DN cells consist of
15 DN1, 2 DN2, and
40 DN3. DN1 and DN2 clusters are composed of middle-sized neurons that are located posterior in the dorsal superior brain, whereas the DN3 cluster contains rather small cells that lie in a very lateral position of the dorsal brain. Because the clock proteins are located predominantly in nuclei and cell bodies of neurons, immunolabeling with anti-PER and anti-TIM could not reveal the morphology of those neurons. This is, however, necessary to understand the neuronal functioning of clock gene-expressing cells. Partial help came from an antiserum against a neuropeptide—the pigment-dispersing factor (PDF)—that is present in the l-LNv cluster and in four cells of the s-LNv cluster (Helfrich-Fo¨ rster, 1995; Kaneko et al., 1997). Arborizations of the other neurons remained unknown until reporter genes were used to label their neurites and dendrites.

Reporter Gene Expression in Clock Neurons

A molecular–genetic technique called the UAS-GAL4 expression system allows the expression of any cloned gene under the control of a specific promoter (Brand and Perrimon, 1993). It uses the yeast transcription factor GAL4, which is put under control of the desired promoter (e.g., the per or tim promoter), for activating the yeast upstream activating sequence ‘‘UAS’’ fused to the gene wished to be expressed. The gene driven by UAS can be a cell marker gene (reporter) such as green fluorescent protein (GFP) or -galactosidase. As a result, the reporter gene is expressed in all cells in which the per (or tim) promoter is active. Unlike PER or TIM, the reporter protein is not restricted to cell bodies but diffuses into the entire neuron and thus labels its arborizations. Reporter proteins such as GFP and -galactosidase have the additional advantage of being very stable. They are thus visible throughout the circadian cycle, making it even possible to detect PER and TIM in weakly expressing cells that cycle out of phase with the others.

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Fig. 1. Clock gene-expressing neurons in the brain of Drosophila melanogaster. (A) Confocal image showing a frontal view of the left brain hemisphere of adult flies stained with anti-PER around lights on in a LD cycle. PER immunostaining is visible in nuclei of large and small ventral lateral neurons (s-LNv and l-LNv), dorsal lateral neurons (LNd), dorsal neuron 1 (DN1), dorsal neuron 2 (DN2), and dorsal neuron 3 (DN3). (B) Confocal image showing arborizations of all clock gene-expressing neurons in a tim-GAL4; UAS-GFP fly, again in the left brain hemisphere. Arrows point to cells that do not express natural tim. (C) Reconstruction of all clock gene-expressing neurons: aMe–accessory medulla, Ca–Calyx of the mushroom body (MB), CC–central complex, Ey–compound eye, La–lamina, Me– medulla, Oc–ocelli, PI–pars intercerebralis, PL–pars lateralis.

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The UAS-GAL4 reporter technique has been used successfully to trace all clock neurons in the larval brain and to show the spatial distribution of clock genes in peripheral tissues of larvae and adults (Kaneko and Hall, 2000). In adult brains, the tracing of neurons was more difficult because staining was also found in many cells that were not PER and TIM immunoreactive. Some of this ‘‘extra staining’’ was the result of position effects associated with the transgenes’ chromosomal insertion. However, certain extra cells were stained in several of the different transgenic lines generated, indicating real promoter activity of the clock genes in these additional cells. This suggests that sequences downstream the promoter region contribute to the spatial regulation of natural gene expression. Indeed, sequences in the coding region of per appear to be necessary for the full spatial expression pattern and for normal kinetics of per oscillations (Stanewsky et al., 1997a,b). Under certain circumstances, this information in the per coding region alone—without any per promoter—is even sufficient to provoke expression in subsets of the clock neurons (Frisch et al., 1994). Despite the aforementioned difficulties, per and tim reporter proteins combined with PER and TIM immunostainings allowed visualization of the neuronal processes of the LNd, DN1, DN2, and DN3 clusters, additionally to those of the l-LNv and s-LNv clusters in adults (Kaneko and Hall, 2000). The arborization pattern of all cells is shown in Fig. 1 and was described in detail previously (Helfrich-Fo¨ rster, 2003; Kaneko and Hall, 2000). The neurites of all per/tim-expressing neurons largely overlap, suggesting that they are functionally connected. All per/tim-expressing neurons except l-LNv cells send their main projections into the dorsal protocerebrum (Fig. 1A and B). The s-LNv, l-LNv, DN1, and DN3 have additional projections toward the accessory medulla—a small neuropil that was shown to house the circadian clock in other insects (for a review, see Helfrich-Fo¨ rster et al., 1998). l-LNv cells connect furthermore both accessory medullae via fibers in the posterior optic tract and send a network of fibers onto the surface of the second optic neuropil—the medulla. The arborization pattern of the different clock gene-expressing neurons suggests that all provide a common circadian output signal that is transferred to the dorsal protocerebrum and, via the l-LNv cells, perhaps also into the optic lobe. The dorsal protocerebrum has connections to most sites of the brain and furthermore houses the neurosecretory system of the fly. Thus, circadian signals arising from the entity of the clock neurons may be transferred electrically and/or via humoral pathways to the effector organs.

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Reporter gene expression was also used to demonstrate the cycling of per and tim expression for several days in vivo. For that purpose, the promoter region of per or tim was fused to the luciferase gene of the firefly (Brandes et al., 1996). In contrast to GFP and -galactosidase, firefly luciferase has a very short live time and enables real-time reporting of gene expression. When fed with luciferin, the living fly shows a cyclic luminescence, which can be recorded with a sensitive scintillation counter. Notably, this recordable luminescence rhythm stems from the compound eyes and other peripheral oscillators, whereas the cycling of the relative few neurons in the brain is not visible in the luminescence output. These peripheral oscillators showed a prominent luciferase cycling under light–dark conditions (Brandes et al., 1996). Even cultured isolated parts of the fly did so, indicating that probably each clock gene-expressing cell contains a light-sensitive autonomous oscillator (Plautz et al., 1997). However, the luciferase cycling dampened more or less rapidly after transfer into constant conditions. The latter observation speaks against a role of the peripheral oscillators in driving behavioral rhythmicity that continues for weeks in darkness. The cycling luminescence in the neurons became visible only after restricting the luciferase reporter to these cells. This was possible with the aforementioned technique using just the spatial information for per expression present within the per gene itself. The promotorless per gene was fused to the luciferase gene and inserted into the fly genome. Several transgenic lines were found with luciferase expression restricted to the DN (Veleri et al., 2003). These flies showed a stable and undampened luciferase cycling, which stands in clear contrast to the cycling of the peripheral cells and shows unequivocally that at least some clock neurons have the capability to drive rhythmic behavior for weeks (see later). In addition to revealing per and tim cycling in vivo, the luciferase technique was also used successfully to screen for new genes involved in the molecular machinery of the circadian clock. Chemical or p-element-based mutagenesis was performed on the per luciferase reporter strain, resulting in the isolation of several mutations that altered luciferase cycling (Stanewsky et al., 1998; Stempfl et al., 2002). So far, only mutants could be detected that alter per cycling in the peripheral oscillators, but the same techniques could be applied to the strain that expresses the luciferase gene only in specific neurons. Such a screen might identify genes that are specifically involved in rhythm generation in clock neurons.

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Genetic Manipulations That Identified Clock Neurons Acting as Circadian Pacemakers for Behavioral Rhythmicity

Although perhaps all clock neurons contain undampened circadian oscillators, not all of them are equally important for maintaining behavioral rhythmicity under constant conditions. This was revealed by elimination of specific neurons through cell death genes or mutation and genetic manipulations of the clock genes in the different neurons. All these studies showed that the three LN groups are more important for driving behavioral rhythmicity under constant conditions than the three DN groups. The three DN groups contribute, however, to rhythmic behavior and can mediate rather normal rhythmicity under light–dark conditions in the absence of functional LN groups. One option to find out the role of the different clusters of clock neurons was the restriction of PER expression to certain cell groups. When the promotorless per luciferase construct (see earlier discussion) was expressed in the three DN clusters of per0 mutants, activity became normal under LD conditions but remained arrythmic under DD conditions (Veleri et al., 2003). When a similar construct (just without the luciferase gene) was expressed solely in the three LN clusters, the activity rhythm of per0 mutants was rescued under LD and DD conditions (Frisch et al., 1994). Studies with mutants showed the same result: disco mutants that retain per cycling in the DN clusters but lack the three LN clusters were more or less normally rhythmic under LD conditions but arrhythmic under DD conditions (Dushay et al., 1989; Hardin et al., 1992; Helfrich-Fo¨ rster, 1998). However, per0/wild-type mosaics with per expression in LN clusters showed robust activity rhythms under both conditions (Ewer et al., 1992). Nevertheless, DN cells contribute to the control of rhythmic activity under DD conditions, as disco mutants show residual rhythms during the first days in constant conditions (Blanchardon et al., 2001; Helfrich-Fo¨ rster, 1998) and transgenic flies with per only in the LN cells have rhythms with lower power and longer period than wild-type flies (Frisch et al., 1994). Although LN cells appear to be the main circadian pacemakers in the fruit fly, it is not clear whether all three LN clusters (LNd, s-LNvS, l-LNv) are equally essential. An important step toward testing the role of the three cell clusters in the circadian system was the cloning of the pdf gene that is only expressed in the l-LNv and s-LNv clusters (Park and Hall, 1998). These two cell clusters could now be manipulated specifically, e.g., by expression of channel, toxin, cell death, or apoptosis genes under control of the pdf promotor. Killing the cells through cell death genes left the flies normally rhythmic under light–dark cycles, but rendered them arrhythmic several days after transfer into constant darkness (Renn et al., 1999). The same

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behavior occurred in a pdf-null mutant (Renn et al., 1999). Electrical silencing of the cells by expression of a permanently open Kþ channel also had little influence on the rhythmicity under light–dark cycles, but again, most flies became arrhythmic under constant darkness (Nitabach et al., 2002). In summary, ablation of the PDF-containing LNv had milder effects on rhythmic behavior than ablation of the LNv and LNd (in disco mutants). This shows that the LNd are as important as the LNv for the control of rhythmicity under DD conditions. Another option to investigate the role of different cell clusters is the overexpression of clock genes in specific clock cells in a wild-type background. This should disrupt the molecular feedback loop specifically in overexpressing cells. Indeed, extensive overexpression of per in the photoreceptor cells of the compound eyes was shown to stop the clock in this tissue, but not in the other per-expressing cells (Cheng and Hardin, 1998); thus, behavioral rhythmicity was undisturbed. However, when clock genes (done for per, tim, vri) were overexpressed in all clock gene-expressing cells (neurons and photoreceptors) using the per or tim promotor as driver, behavior became arrhythmic under DD conditions (Blau and Young, 1999; Kaneko et al., 2000; Yang and Sehgal, 2001). The behavior under LD conditions was only studied for per overexpression and was changed significantly, although the flies became not totally arrhythmic (Kaneko et al., 2000). When per was overexpressed in the LN clusters (strongly in the s-LNv and l-LNv and weaker in the LNd), behavior became arrhythmic under DD, but remained normal under LL conditions (Blanchardon et al., 2001), again indicating that LN clusters are most important for behavioral rhythmicity under DD. Restricting per overexpression to the LNv clusters alone did not result in arrhythmicity for adult behavior (Yang and Sehgal, 2001) but disrupted the eclosion rhythm of the flies out of their pupae (Myers et al., 2003). In summary, the mentioned results indicate that the circadian clock controlling behavioral rhythmicity is composed of a network of clock geneexpressing neurons that interact in a distinct way. Under the clock neurons a clear hierarchy is visible, with LN groups standing in the first place and DN groups in the second. Of the LN groups, LNd and PDF-expressing LNv appear similarly important in controlling rhythmic activity under DD conditions. The specific roles of the s-LNv and l-LNv subclusters, as well as those of the three DN clusters, remain to be revealed. A promising technique might be the combination of the GAL4 system with the GAL80 system (Duffy, 2002). GAL80 represses GAL4-driven gene expression. Thus, two different promoters can be combined: one driving GAL4 in several cell clusters and the other driving GAL80 in certain subclusters. A suited promoter could, for example, drive GAL4 in all three LN clusters

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and another GAL80 only in the two LNv clusters of the same fly; then GAL4 will only be activated in the LNd. Consequently, the LNd could be eliminated specifically by expression of a cell death gene under the control of GAL4. Ectopic Expression of Clock and Clock-Related Genes

The UAS-GAL4 system and related techniques were also used to drive clock gene expression in cells that normally do not express any clock gene in order to learn more about specific clock factors. In 1995, Vosshall and Young expressed the per gene under control of the glass promoter in a per0 mutant background. Interestingly, they could rescue the arrhythmic behavior of the flies, suggesting that glass is expressed in clock neurons or that ectopic cells can overtake the role of the clock cells. Both turned out to be true: The glass gene is expressed in the DN1 cluster and in a group of cells in the lateral brain close to the LNd cluster (Klarsfeld et al., 2004; Vosshall and Young, 1995). The latter cells even have similar projections to that of the LNd, but were clearly not identical with them (Klarsfeld et al., 2004). Since the DN1 group was recently found to be the only DN cluster without self-sustained PER oscillations and thus not suited to control the activity rhythm under DD conditions (Klarsfeld et al., 2004; Veleri et al., 2003), it is more likely that the LNd-like cluster can overtake the role of the LNd and drive rhythmic behavior. This is a special case of ectopic clock gene expression where the ectopic cells were similar to the naturally per-expressing neurons. However, in most cases, ectopic clock gene expression is quite different to the natural expression and results in a change or disruption of behavioral rhythmicity. The kind of change and degree of disruption may tell something about the role of the ectopically expressed clock factors, as was shown for Clk and PDF. Clk is a special clock gene because it is also involved in development and its lack (e.g., in the clk JRK mutant) results in aberrant arborizations of certain clock neurons (Park et al., 2000) in addition to behavioral arrhythmicity. Interestingly, ectopic Clk expression could induce molecular clocks in cells that normally do not express any clock gene, demonstrating again the role of Clk in development, perhaps as a kind of master gene (Zhao et al., 2003). The induced ectopic clocks appeared functional and interfered with the natural clock cells, leading to disturbed behavioral rhythms. Unlike Clk, PDF is not involved in the mechanism of the core clock, but acts as a neuropeptide transmitter in the LNv. PDF release is under clock control, as PDF immunoreactivity varies cyclically in the terminals of the s-LNv cells, the cycle has a short period in pers mutants, and is arrhythmic in per0 and tim0 flies (Park et al.,

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2000). Furthermore, PDF levels were extremely low in cyc01, clk JRK mutants, and vrille-overexpressing flies (Blau and Young, 1999; Park et al., 2000). When PDF is expressed ectopically in the dorsal brain, the flies are hyperactive, either lengthen period or show splitting in several free-running components, and often become arrhythmic (Helfrich-Fo¨ rster et al., 2000). This behavior might be caused by elevated PDF levels in the dorsal brain, which interfere with the naturally released PDF from the s-LNv terminals, putatively producing conflicting signals. Both findings are consistent with a role of PDF as a mediator for circadian signals to downstream neurons. Nevertheless, PDF might also serve as a coupling factor between the different clock neurons (Peng et al., 2003). Then, it may feedback indirectly or even directly on the molecular cycle of these neurons, influencing their period. This would explain the lengthened period of flies that express PDF ectopically. It is furthermore consistent with a short-period rhythm found in pdf 0 mutants for several days before the flies become arrhythmic (Renn et al., 1999). Role of Peripheral Oscillators

From the previously mentioned results, it became quite clear that the peripheral oscillators are not important for behavioral rhythmicity. So what is the physiological function of these peripheral light-entrainable autonomous clocks? Some answers were found for the antennae, compound eyes, and in the male gonads. The antennae show a circadian rhythm in the electroantennogram, which is likely to be responsible for circadian differences in sensitivity to olfactory stimuli (Krishnan et al., 1999). A similar role for the circadian clock has also been proposed in the modulation of the photic sensitivity of the compound eye (Chen et al., 1992). In addition, the male gonads’ spermatophore production and sperm mobility was found to occur in a rhythmic manner, and the possession of an intact rhythm in these functions significantly increases the reproductive fitness (Beaver et al., 2002). It is quite imaginable that coordinated rhythms in gut, malpighian tubules, and fat body have similar adaptive advantages for digestion, detoxification, and fat metabolism. In mammals, analogous peripheral oscillators are also present, showing a remarkable independence from the master clock in the brain (reviewed by Schibler and Sassone-Corsi, 2002). In contrast to flies, these are not light sensitive, but are sensitive to chemical cues or to temperature cycles (Balsalobre et al., 2000; Brown et al., 2002; Schibler et al., 2003). Furthermore, the master clock in the brain of mammals controls the peripheral oscillators hierarchically by humoral and/or neuronal signals. Only if the master clock is lesioned is circadian gene expression dampened in the peripheral tissues

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(Reppert and Weaver, 2002). In Drosophila, the peripheral clocks dampen even in vivo (thus with normal connection to the master clock) as soon as the animals are exposed to constant conditions (Plautz et al., 1997). Thus, the peripheral clocks of Drosophila are not controlled by the master clock in the brain, but are governed directly by the external light–dark cycle. Dampening is most likely caused by a desynchrony of the peripheral oscillators due to the lack of a synchronizing light input. Concluding Remarks

Paired with classical immunocytochemistry, genetic manipulations have been most successful in unraveling the main network of the circadian clock of Drosophila. Peripheral oscillators with remarkable independence from the brain were identified using reporter genes. These peripheral oscillators appear to govern rhythms in receptor sensitivity as well as different metabolic and physiological parameters. Behavioral rhythmicity, however, depends on certain pacemaker neurons in the brain, as was revealed by generating genetic mosaics, mutants, and manipulation of clock gene expression. Reporter genes unraveled a neuronal network formed by all clock gene-expressing neurons in the brain, enabling mutual interaction between them. Manipulating certain clusters using the UASGAL4 system revealed a clear hierarchy in this network. LN clusters appear more important for behavioral rhythmicity under constant conditions than DN clusters. The specific roles of the three LN and DN subclusters have yet to be revealed. Combination of the UAS-GAL4 system with the GAL80 system might be a promising technique to further dissect the circadian pacemaker system of the fly. References Balsalobre, A., Brown, S. A., Marcacci, L., Tronche, F., Kellendonk, C., Reichardt, H. M., Schutz, G., and Schibler, U. (2000). Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 289, 2344–2347. Beaver, L. M., Gvakharia, B. O., Vollintine, T. S., Hege, D. M., Stanewsky, R., and Giebultowicz, J. M. (2002). Loss of circadian clock function decreases reproductive fitness in males of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 99, 2134–2139. Blanchardon, E., Grima, B., Klarsfeld, A., Chelot, E., Hardin, P. E., Pre´ at, T., and Rouyer, F. (2001). Defining the role of Drosophila lateral neurons in the control of activity and eclosion rhythms by targeted genetic ablation and PERIOD overexpression. Eur. J. Neurosci. 13, 871–888. Blau, J., and Young, M. W. (1999). Cycling vrille expression is required for a functional Drosophila clock. Cell 99, 661–671. Brand, A. H., and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.

[21]

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Brandes, C., Plautz, J. D., Stanewsky, R., Jamison, C. F., Straume, M., Wood, K. V., Kay, S. A., and Hall, J. C. (1996). Novel features of Drosophila period transcription revealed by realtime luciferase reporting. Neuron 16, 687–692. Brown, S. A., Zumbrunn, G., Fleury-Olela, F., Preitner, N., and Schibler, U. (2002). Rhythms of mammalian body temperature can sustain peripheral circadian clocks. Curr. Biol. 12, 1574–1583. Chen, D.-M., Christianson, J. S., Sapp, R. J., and Stark, W. S. (1992). Visual receptor cycle in normal and period mutant Drosophila: Microspectrophotometry, electrophysiology, and ultrastructural morphometry. Vis. Neurosci. 9, 125–135. Cheng, Y., and Hardin, P. E. (1998). Drosophila photoreceptors contain an autonomous circadian oscillator that can function without period mRNA cycling. J. Neurosci. 18, 741–750. Duffy, J. B. (2002). GAL4 system in Drosophila: A fly geneticist’s Swiss army knife. Genesis 34, 1–15. Dushay, M. S., Rosbash, M., and Hall, J. C. (1989). The disconnected visual system mutations in Drosophila drastically disrupt circadian rhythms. J. Biol. Rhythms 4, 1–27. Ewer, J., Frisch, B., Hamblen-Coyle, M. J., Rosbash, M., and Hall, J. C. (1992). Expression of the period clock gene within different cell types in the brain of Drosophila adults and mosaic analysis of these cells’ influence on circadian behavioral rhythms. J. Neurosci. 12, 3321–3349. Frisch, B., Hardin, P. E., Hamblen-Coyle, M. J., Rosbash, M., and Hall, J. C. (1994). A promoterless period gene mediates behavioral rhythmicity and cyclical per expression in a restricted subset of the Drosophila nervous system. Neuron 12, 555–570. Hall, J. C. (1998). Genetics of biological rhythms in Drosophila. Adv. Genet. 38, 135–184. Hardin, P. E., Hall, J. C., and Rosbash, M. (1992). Behavioral and molecular analyses suggest that circadian output is disrupted by disconnected mutants in D. melanogaster. EMBO J. 11, 1–6. Helfrich-Fo¨ rster, C. (1995). The period clock gene is expressed in CNS neurons which also produce a neuropeptide that reveals the projections of circadian pacemaker cells within the brain of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 92, 612–616. Helfrich-Fo¨ rster, C. (1998). Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: A brain-behavioral study of disconnected mutants. J. Comp. Physiol. A 182, 435–453. Helfrich-Fo¨ rster, C. (2002). The circadian system of Drosophila melanogaster and its light input pathways. Zoology 105, 297–312. Helfrich-Fo¨ rster, C. (2003). The neuroarchitecture of the circadian clock in the Drosophila brain. Microsc. Res. Tech. 62, 94–102. Helfrich-Fo¨ rster, C., Stengl, M., and Homberg, U. (1998). Organization of the circadian system in insects. Chronobiol. Int. 15, 567–594. Helfrich-Fo¨ rster, C., Ta¨ uber, M., Park, J. H., Mu¨ hlig-Versen, M., Schneuwly, S., and Hofbauer, A. (2000). Ectopic expression of the neuropeptide pigment-dispersing factor alters behavioral rhythms in Drosophila melanogaster. J. Neurosci. 20, 3339–3353. Kaneko, M., and Hall, J. C. (2000). Neuroanatomy of cells expressing clock genes in Drosophila: Transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections. J. Comp. Neurol. 422, 66–94. Kaneko, M., Helfrich-Fo¨ rster, C., and Hall, J. C. (1997). Spatial and temporal expression of the period and timeless genes in the developing nervous system of Drosophila: Newly identified pacemaker candidates and novel features of clock gene product cycling. J. Neurosci. 17, 6745–6760. Kaneko, M., Park, J. H., Cheng, Y., Hardin, P. E., and Hall, J. C. (2000). Disruption of synaptic transmission or clock-gene-product oscillations in circadian pacemaker cells of Drosophila cause abnormal behavioral rhythms. J. Neurobiol. 43, 207–233.

450

anatomical representation of neural clocks

[21]

Klarsfeld, A., Malpel, S., Michard-Vanhee´ , C., Picot, M., Che´ lot, E., and Rouyer, F. (2004). Novel features of cryptochrome-mediated photoreception in the brain circadian clock of Drosophila. J. Neurosci. 24, 1468–1477. Krishnan, B., Dryer, S. E., and Hardin, P. E. (1999). Circadian rhythms in olfactory responses of Drosophila melanogaster. Nature 400, 375–378. Myers, E. M., Yu, J., and Sehgal, A. (2003). Circadian control of eclosion: Interaction between a central and peripheral clock in Drosophila melanogaster. Curr. Biol. 13, 526–533. Nitabach, M. N., Blau, J., and Holmes, T. C. (2002). Electrical silencing of Drosophila pacemaker neurons stops the free-running circadian clock. Cell 109, 485–495. Park, J. H., and Hall, J. C. (1998). Isolation and chronobiological analysis of a neuropeptide pigment-dispersing factor gene in Drosophila melanogaster. J. Biol. Rhythms 13, 219–228. Park, J. H., Helfrich-Fo¨ rster, C., Lee, G., Liu, L., Rosbash, M., and Hall, J. C. (2000). Differential regulation of circadian pacemaker output by separate clock genes in Drosophila. Proc. Natl. Acad. Sci. USA 97, 3608–3613. Peng, Y., Stoleru, D., Levine, J. D., Hall, J. C., and Rosbash, M. (2003). Drosophila freerunning rhythms require intercellular communication. PLoS Biol. 1, E13. Plautz, J. D., Kaneko, M., Hall, J. C., and Kay, S. A. (1997). Independent photoreceptive circadian clocks throughout Drosophila. Science 278, 1632–1635. Renn, S. C. P., Park, J. H., Rosbash, M., Hall, J. C., and Taghert, P. H. (1999). A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 99, 791–802. Reppert, S. M., and Weaver, D. R. (2002). Coordination of circadian timing in mammals. Nature 418, 935–941. Schibler, U., Ripperger, J., and Brown, S. A. (2003). Peripheral circadian oscillators in mammals: Time and food. J. Biol. Rhythms 18, 250–260. Schibler, U., and Sassone-Corsi, P. (2002). A web of circadian pacemakers. Cell 111, 919–922. Shafer, O. T., Rosbash, M., and Truman, J. W. (2002). Sequential nuclear accumulation of the clock proteins period and timeless in the pacemaker neurons of Drosophila melanogaster. J. Neurosci. 22, 5946–5954. Siwicki, K. K., Eastman, C., Petersen, G., Rosbash, M., and Hall, J. C. (1988). Antibodies to the period gene product of Drosophila reveal diverse tissue distribution and rhythm changes in the visual system. Neuron 1, 141–150. Stanewsky, R., Frisch, B., Brandes, C., Hamblen-Coyle, M., Rosbash, M., and Hall, J. C. (1997a). Temporal and spatial expression patterns of transgenes containing increasing amounts of the Drosophila clock gene period and a lacZ reporter: Mapping elements of the PER protein involved in circadian cycling. J. Neurosci. 17, 676–696. Stanewsky, R., Jamison, C. F., Plautz, J. D., Kay, S. A., and Hall, J. C. (1997b). Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila. EMBO J. 16, 5006–5018. Stanewsky, R., Kaneko, M., Emery, P., Beretta, B., Wager-Smith, K., Kay, S. A., Rosbash, M., and Hall, J. C. (1998). The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95, 681–692. Stempfl, T., Vogel, M., Szabo, G., Wu¨ lbeck, C., Liu, J., Hall, J. C., and Stanewsky, R. (2002). Identification of circadian-clock-regulated enhancers and genes of Drosophila melanogaster by transposon mobilization and luciferase reporting of cyclical gene expression. Genetics 160, 571–593. Veleri, S., Brandes, C., Helfrich-Fo¨ rster, C., Hall, J. C., and Stanewsky, R. (2003). A selfsustaining, light-entrainable circadian oscillator in the Drosophila brain. Curr. Biol. 13, 1758–1767.

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Vosshall, L. B., and Young, M. W. (1995). Circadian rhythms in Drosophila can be driven by period expression in a restricted group of central brain cells. Neuron 15, 345–360. Yang, Z., and Sehgal, A. (2001). Role of molecular oscillations in generating behavioral rhythms in Drosophila. Neuron 29, 453–467. Zerr, D. M., Hall, J. C., Rosbash, M., and Siwicki, K. K. (1990). Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drosophila. J. Neurosci. 10, 2749–2762. Zhao, J., Kilman, V. L., Keegan, K. P., Peng, Y., Emery, P., Rosbash, M., and Allada, R. (2003). Drosophila clock can generate ectopic circadian clocks. Cell 113, 755–766.

[22] The Suprachiasmatic Nucleus is a Functionally Heterogeneous Timekeeping Organ By Rae Silver and William J. Schwartz Abstract

Ever since the locus of the brain clock in the suprachiasmatic nucleus (SCN) was first described, methods available have both enabled and encumbered our understanding of its nature at the level of the cell, the tissue, and the animal. A combination of in vitro and in vivo approaches has shown that the SCN is a complex heterogeneous neuronal network. The nucleus is composed of cells that are retinorecipient and reset by photic input; those that are reset by nonphotic inputs; slave oscillators that are rhythmic only in the presence of the retinohypothalamic tract; endogenously rhythmic cells, with diverse period, phase, and amplitude responses; and cells that do not oscillate, at least on some measures. Network aspects of SCN organization are currently being revealed, but mapping these properties onto cellular characteristics of electrical responses and patterns of gene expression are in early stages. While previous mathematical models focused on properties of uniform coupled oscillators, newer models of the SCN as a brain clock now incorporate oscillator and gated, nonoscillator elements.

The Brain’s Clock as a Construct

The function of the suprachiasmatic nucleus (SCN) was discovered during the era that identified the hypothalamus as the site of several brain ‘‘centers’’ governing homeostatically regulated behaviors. Ablation of the lateral hypothalamus resulted in a significant reduction of eating behavior

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